MOLECULAR CLONING, EUKARYOTIC EXPRESSION AND FUNCTION STUDY OF FTR56 FROM DANIO RERIO

To understand the role of zebrafish finTRIM in antiviral innate immunity, the zebrafish ftr56 gene was cloned and analyzed for its effect on the proliferation of spring viremia of carp virus (SVCV). Primers were designed according to the zebrafish FTR56 sequence. The FTR56 CDS region was amplified by PCR and ligated into the eukaryotic expression vector pcDNA4.0-His to construct the eukaryotic expression plasmid pcDNA4.0-FTR56-His and conducted bioinformatics analysis. Real-time quantitative PCR (qRT-PCR) was used to detect the expression of FTR56 mRNA in SVCV-infected zebrafish embryo fibroblasts (ZF4). Phylogenetic tree analysis showed that the zebrafish FTR56 was individually clustered. The amino acid sequence alignment showed that the similarites with TRIM56 of chimpanzees, cattle and mice were 22%—23%. The FTR56 secondary structure has one RING finger domain, one B-box domain, one coiled-coil region and one B30.0 domain. The FTR56 mRNA level increased significantly at 24h after SVCV infection. After overexpression of FTR56, the mRNA and protein levels of G protein of SVCV reduced significantly at 12h and 24h compared with the control group associated with significantly decreased SVCV titers in the culture supernatant, indicating that FTR56 inhibited SVCV proliferation. This study provide a reference for further revealing the immunoregulatory mechanism of finTRIM in fish viral diseases.